Giant axon formation in mice lacking Kell, XK, or Kell and XK: animal models of McLeod neuroacanthocytosis syndrome

Abstract

McLeod neuroacanthocytosis syndrome (MLS) is a rare X-linked multisystem disease caused by XK gene mutations and characterized by hematological and neurological abnormalities. XK, a putative membrane transporter, is expressed ubiquitously and is covalently linked to Kell, an endothelin-3-converting enzyme (ECE-3). Absence of XK results in reduction of Kell at sites where both proteins are coexpressed. To elucidate the functional roles of XK, Kell, and the XK-Kell complex associated with pathogenesis in MLS, we studied the pathology of the spinal cord, anterior roots, sciatic nerve, and skeletal muscle from knockout mouse models, using Kel(-/-), Xk(-/-), Kel(-/-)Xk(-/-), and wild-type mice aged 6 to 18 months. A striking finding was that giant axons were frequently associated with paranodal demyelination. The pathology suggests probable anterograde progression from the spinal cord to the sciatic nerve. The neuropathological abnormalities were found in all three genotypes, but were more marked in the double-knockout Kel(-/-)Xk(-/-) mice than in either Kel(-/-) or Xk(-/-) mice. Skeletal muscles from Xk(-/-) and Kel(-/-)Xk(-/-) mice showed mild abnormalities, but those from Kel(-/-) mice were similar to the wild type. The more marked neuropathological abnormalities in Kel(-/-)Xk(-/-) mice suggest a possible functional association between XK and Kell in nonerythroid tissues.

Figure 1

Giant axons (arrows) in mouse sciatic nerve. A: Transverse section (WT). B: Large demyelinated axon from transverse section (Kel^−/−Xk^−/−). C: Large axons with thin myelin sheath from longitudinal section (Xk^−/−). D: A portion of an internode with partial demyelination from a longitudinal section (Xk^−/−). E: Large axon with disrupted myelin sheath and unevenly distributed osmium-dense material (Kel^−/−Xk^−/−). F: Atrophic axon with thick myelin sheath (Xk^−/−). Sections are 1 μm thick. Toluidine Blue stain. Original magnification, ×1000.

Figure 2

Giant axons (arrows) in the anterior column and ventral root of mouse spinal cord. A: Transverse section of the anterior column (from WT mice). B: Several large axons with relatively thin myelin sheaths on a transverse section of anterior column (Kel^−/−). C and D: Irregular thickness of myelin sheath with paranodal demyelination and partial detachment of myelin leaflets in a longitudinal section of the ventral root [Kel^−/−Xk^−/− (C) and Kel^−/− (D)]. E: Enlarged axon packed with osmium-dense particles and vacuoles with partial disruption of myelin sheath from a longitudinal section of the ventral root (Kel^−/−). F: Paranodal demyelination from a longitudinal section of the ventral root of exercised mice (Xk^−/−). Sections are 1 μm thick. Toluidine Blue stain. Original magnification, ×1000.

Figure 3

Ultrastructural details of giant axons in spinal cord and sciatic nerve. A: Giant axon with an extremely attenuated myelin sheath in the anterior column of the spinal cord (Kel^−/−Xk^−/−). B: Giant axon with an extremely attenuated myelin sheath in the sciatic nerve (Xk^−/−). C: Giant axon with irregular accumulation of NFs and apparent absence of other organelles in the sciatic nerve (Xk^−/−). D: Giant axon with a thin myelin sheath containing irregularly arranged NFs and sparsely scattered microtubules in the anterior column of the spinal cord (Kel^−/−Xk^−/−). E: Segregation of microtubules from NFs on a cross section of the sciatic nerve (Kel^−/−Xk^−/−). F: Irregularly dispersed microtubules in the anterior column of the spinal cord (Kel^−/−). G: Accumulation of dense lamellar bodies and membranous vesicles indicating axonal degeneration in the anterior column of the spinal cord (Kel^−/−Xk^−/−). H: Membranous structures involving the adaxon of a Schwann cell in the anterior column of the spinal cord (Kel^−/−). I: Atrophic axon with contracted axon surrounded by a thick myelin sheath containing osmium-dense particles in the sciatic nerve of Kel^−/−Xk^−/− mice. Symbols: opposing arrows, thin or absent MS; short black arrow or black star, NF; long black arrow, MV; black arrowhead or white star, MT; white arrow, DLB. Sections are 80 nm thick. Scale bars: 2 μm (A, B, and I); 500 nm (C–H). DLB, dense lamellar body; M, mitochondria; MS, myelin sheath; MT, microtubule; MV, membranous vesicles; SciN, sciatic nerve; SpAC, anterior column of the spinal cord.

Figure 4

Skeletal muscle abnormalities. A: WT quadriceps femoris. B and C: Triceps brachii with angulated fibers (black arrows) of Kel^−/−Xk^−/− (B) and Xk^−/− (C) mice. D: Quadriceps femoris of Xk^−/− exercised mice with angulated fibers (black arrows) and internalized nuclei (white arrow). H&E stain. Sections are 5 μm thick. Original magnification: ×200 (A, B, and D); ×400 (C).